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Purpose: After observing variation in the expression of the housekeeping gene B2M in cells of the urinary sediment during a study of candidate genes potentially involved in diabetic kidney disease (DKD), we hypothesized that B2M mRNA expression in the urinary sediment could reflect the presence of DKD., Methods: qPCR was used to quantify B2M mRNA expression in cells of the urinary sediment of 51 type 1 diabetes (T1D) patients (61% women, 33.5 [27.0-39.7] years old, with diabetes duration of 21.0 [15.0-28.0] years and HbA1c of 8.2% [7.3-8.9]; median [interquartile interval]) sorted according to the diabetic nephropathy (DN) stages; 8 focal segmental glomerulosclerosis (FSGS) patients and 10 healthy controls. B2M mRNA expression was also evaluated in human embryonic kidney epithelium-like (HEK-293) cells exposed to 25mM glucose and to albumin in order to mimic, respectively, a diabetic and a proteinuric milieu., Results: No differences were found in B2M mRNA expression among healthy controls, FSGS and T1D patients. Nonetheless B2M mRNA expression was higher in the group composed by T1D patients with incipient or overt DN combined with FSGS patients versus T1D patients without DN combined with healthy controls (P=0.0007). B2M mRNA expression was higher in T1D patients with incipient or overt DN versus without DN (P=0.03). B2M mRNA expression positively correlated with albuminuria in the overall T1D population (r=0.43; P=0.01) and negatively correlated with estimated glomerular filtration rate in male T1D patients (r=- 0.57; P=0.01). Increased B2M expression was observed in HEK-293 cells exposed to 25mM glucose and to albumin., Conclusions: Β2M mRNA expression in cells of the urinary sediment is higher in T1D patients with DKD and in patients with FSGS in comparison to healthy subjects, maybe reflecting a tubulointerstitial injury promoted by albumin. Given the proinflammatory nature of B2M, we suggest that this protein contributes to diabetic (and possibly, to non-diabetic) tubulopathy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Aims: The fetal programming hypothesis suggests that intrauterine stimuli can induce metabolic changes in offspring, increasing the disease risk in adulthood. Periodontal disease may enhance serum cytokine levels. Cytokines such as tumor necrosis factor-alpha (TNF-α) have been associated with reduced glucose transporter type 4 (GLUT4) expression, decreased protein kinase B (Akt) phosphorylation, and insulin resistance. This study aimed to evaluate GLUT4 content, and Akt serine phosphorylation status in the gastrocnemius skeletal muscle (GSM), glycemia, insulinemia and change in body weight in offspring of rats with periodontal disease., Main Methods: Female Wistar rats were distributed into a control group (CN) and an experimental periodontal disease group (PD), in which a ligature was placed around the mandibular first molars. Seven days after ligature placement, both groups were mated with normal male rats. The ligatures remained throughout pregnancy until weaning, after which the male offspring were distributed into groups: CN-o, control rat offspring; and PD-o, periodontal disease rat offspring. The body weight from 0 to 75days of age was measured. At 75days, the glycemia, insulinemia, TNF-α levels, Akt serine phosphorylation, and GLUT4 content in the GSM were measured in the offspring., Key Findings: The PD-o group showed a low birth weight (LBW), unchanged glycemia, increased insulinemia, insulin resistance, increased TNF-α levels, decreased Akt serine phosphorylation status, and reduced GLUT4 content in the plasma membrane and translocation index after insulin stimulation., Significance: Maternal periodontal disease causes LBW, insulin resistance, and alterations in the final stage of insulin signaling in the GSM of adult offspring., (Copyright © 2016 Elsevier Inc. All rights reserved.)

High glucose concentration in the airway surface liquid (ASL) is an important feature of diabetes that predisposes to respiratory infections. We investigated the role of alveolar epithelial SGLT1 activity on ASL glucose concentration and bacterial proliferation. Non-diabetic and diabetic rats were intranasally treated with saline, isoproterenol (to increase SGLT1 activity) or phlorizin (to decrease SGLT1 activity); 2 hours later, glucose concentration and bacterial proliferation (methicillin-resistant Sthaphylococcus aureus, MRSA and Pseudomonas aeruginosa, P. aeruginosa) were analyzed in bronchoalveolar lavage (BAL); and alveolar SGLT1 was analyzed by immunohistochemistry. BAL glucose concentration and bacterial proliferation increased in diabetic animals: isoproterenol stimulated SGLT1 migration to luminal membrane, and reduced (50%) the BAL glucose concentration; whereas phlorizin increased the BAL glucose concentration (100%). These regulations were accompanied by parallel changes of in vitro MRSA and P. aeruginosa proliferation in BAL (r = 0.9651 and r = 0.9613, respectively, Pearson correlation). The same regulations were observed in in vivo P. aeruginosa proliferation. In summary, the results indicate a relationship among SGLT1 activity, ASL glucose concentration and pulmonary bacterial proliferation. Besides, the study highlights that, in situations of pulmonary infection risk, such as in diabetic subjects, increased SGLT1 activity may prevent bacterial proliferation whereas decreased SGLT1 activity can exacerbate it.

Chrysobalanus icaco L. is a medicinal plant present in the Brazilian coastline and known for its hypoglicemic and antioxidant properties. Here, we assessed the beneficial metabolic effects of the aqueous extract of C. icaco (AECI) leaves in diet-induced obese mice. Swiss mice were fed standard chow (SC used as controls) or high-fat diet (HFD) to induce obesity. After 10 weeks, mice on each diet were divided into two groups with one group used as control while the other group treated with AECI for 4 weeks resulting in four groups of mice: SC; SC treated with AECI (SC + AECI); HFD; and HFD treated with AECI (HFD + AECI). AECI was administered drinking water at about 200 mg/kg. AECI was able to normalize insulin (13,682 ± 1090 vs. 9828 ± 485 AU, P < .05) and fasting blood glucose (192.8 ± 14.2 vs. 132.3 ± 6.4 mg/dL, P < .05) and inhibit weight gain (39 ± 5.7%) and fat storage in liver (72.60 ± 3.83%, P < .0001), despite the high-fat intake. These findings reinforce the use of AECI in hyperglycemia and highlight the potential extract's effect in preventing weight gain and fat accumulation in liver of diet-induced obese mice.

Aim: To assess the renal effects of chronic exposure to advanced glycation end-products (AGEs) in the absence of diabetes and the potential impact of concomitant treatment with the antioxidant N-acetyl cysteine (NAC)., Methods: Wistar rats received intraperitoneally 20 mg/kg/day of albumin modified (AlbAGE) or not (AlbC) by advanced glycation for 12 weeks and oral NAC (600mg/L; AlbAGE+NAC and AlbC+NAC, respectively). Biochemical, urinary and renal morphological analyses; carboxymethyl-lysine (CML, an AGE), CD68 (macrophage infiltration), and 4-hydroxynonenal (4-HNE, marker of oxidative stress) immunostaining; intrarenal mRNA expression of genes belonging to pathways related to AGEs (Ager, Ddost, Nfkb1), renin-angiotensin system (Agt, Ren, Ace), fibrosis (Tgfb1, Col4a1), oxidative stress (Nox4, Txnip), and apoptosis (Bax, Bcl2); and reactive oxidative species (ROS) content were performed., Results: AlbAGE significantly increased urine protein-to-creatinine ratio; glomerular area; renal CML content and macrophage infiltration; expression of Ager, Nfkb1, Agt, Ren, Tgfb1, Col4a1, Txnip, Bax/Bcl2 ratio; and 4-HNE and ROS contents. Some of these effects were attenuated by NAC concomitant treatment., Conclusion: Because AGEs are highly consumed in modern diets and implicated in the progression of different kidney diseases, NAC could be a therapeutic intervention to decrease renal damage, considering that long-term restriction of dietary AGEs is difficult to achieve in practice., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

Aims: Thioredoxin interacting protein (TXNIP), an inhibitor of antioxidant thioredoxin (Trx), is upregulated by hyperglycemia and implicated in pathogenesis of diabetes complications. We evaluated mRNA expressions of genes encoding TXNIP and Trx (TXN) in urinary sediment and peripheral blood mononuclear cells (PBMC) of type 1 diabetes (T1D) patients with different degrees of chronic complications., Methods: qPCR was employed to quantify target genes in urinary sediment (n = 55) and PBMC (n = 161) from patients sorted by presence or absence of diabetic nephropathy (DN), retinopathy, peripheral and cardiovascular neuropathy; 26 healthy controls and 13 patients presenting non-diabetic nephropathy (focal and segmental glomerulosclerosis, FSGS) were also included., Results: Regarding the urinary sediment, TXNIP (but not TXN) expression was higher in T1D (p = 0.0023) and FSGS (p = 0.0027) patients versus controls. Expressions of TXNIP and TXN were higher, respectively, in T1D patients with versus without DN (p = 0.032) and in those with estimated glomerular filtration rate (eGFR) < 60 versus ≥60 mL/min/1.73 m(2) (p = 0.008). eGFR negatively correlated with TXNIP (p = 0.04, r = -0.28) and TXN (p = 0.04, r = -0.30) expressions. T1D patients who lost ≥5 mL/min/1.73 m(2) yearly of eGFR presented higher basal TXNIP expression than those who lost <5 mL/min/1.73 m(2) yearly after median follow-up of 24 months. TXNIP (p < 0.0001) and TXN (p = 0.002) expressions in PBMC of T1D patients were significantly higher than in controls but no differences were observed between patients with or without chronic complications., Conclusions: TXNIP and TXN are upregulated in urinary sediment of T1D patients with diabetic kidney disease (DKD), but only TXNIP expression is associated with magnitude of eGFR decline.

Objective: Unequivocal modulation of glycemic homeostasis by chronic beta-adrenergic blockade in diabetes has never been demonstrated. This study investigates the participation of beta-adrenergic system in glycemic control and muscle glucose transporter GLUT4 expression in insulin-treated diabetic rats., Methods: Insulin-treated diabetic Wistar (W) or spontaneously hypertensive rats (SHR) were additionally treated with propranolol, and glycemic homeostasis and expression of some target mRNAs and proteins in soleus and extensor digitorum longus (EDL) muscles were analyzed., Results: Insulin improved glycemic control in both strains. Importantly, in W, propranolol promoted a further improvement in glycemic control, which was accompanied by decreased PKA and Tnf expression, and increased Slc2a4 and GLUT4 in EDL. Those effects were not observed in diabetic-SHR., Discussion: Propranolol-induced decrease in beta-adrenergic activity in skeletal muscles of insulin-treated diabetic Wistar rats increases GLUT4 expression in EDL, improving glycemic control. These outcomes represent a positive effect of nonselective beta-blockade, which might be extended to autonomic neuropathy., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Regular exercise prevents and regresses atherosclerosis by improving lipid metabolism and antioxidant defenses. Exercise ameliorates the reverse cholesterol transport (RCT), an antiatherogenic system that drives cholesterol from arterial macrophages to the liver for excretion into bile and feces. In this study we analyzed the role of aerobic exercise on the in vivo RCT and expression of genes and proteins involved in lipid flux and inflammation in peritoneal macrophages, aortic arch and liver from wild type mice., Methods: Twelve-week-old male mice were divided into sedentary and trained groups. Exercise training was performed in a treadmill (15 m/min, 30 min/day, 5 days/week). Plasma lipids were determined by enzymatic methods and lipoprotein profile by fast protein liquid chromatography. After intraperitoneal injection of J774-macrophages the RCT was assessed by measuring the recovery of (3)H-cholesterol in plasma, feces and liver. The expression of liver receptors was determined by immunoblot, macrophages and aortic mRNAs by qRT-PCR. (14)C-cholesterol efflux mediated by apo A-I and HDL2 and the uptake of (3)H-cholesteryl oleoyl ether ((3)H-COE)-acetylated-LDL were determined in macrophages isolated from sedentary and trained animals 48 h after the last exercise session., Results: Body weight, plasma lipids, lipoprotein profile, glucose and blood pressure were not modified by exercise training. A greater amount of (3)H-cholesterol was recovered in plasma (24 h and 48 h) and liver (48 h) from trained animals in comparison to sedentary. No difference was found in (3)H-cholesterol excreted in feces between trained and sedentary mice. The hepatic expression of scavenger receptor class B type I (SR-BI) and LDL receptor (B-E) was enhanced by exercise. We observed 2.8 and 1.7 fold rise, respectively, in LXR and Cyp7a mRNA in the liver of trained as compared to sedentary mice. Macrophage and aortic expression of genes involved in lipid efflux was not systematically changed by physical exercise. In agreement, (14)C-cholesterol efflux and uptake of (3)H-COE-acetylated-LDL by macrophages was similar between sedentary and trained animals., Conclusion: Aerobic exercise in vivo accelerates the traffic of cholesterol from macrophages to the liver contributing to prevention and regression of atherosclerosis, independently of changes in macrophage and aorta gene expression.

Advanced glycation end products (AGE) are elevated in diabetes mellitus (DM) and predict the development of atherosclerosis. AGE-albumin induces oxidative stress, which is linked to a reduction in ABCA-1 and cholesterol efflux. We characterized the glycation level of human serum albumin (HSA) isolated from poorly controlled DM2 (n = 11) patients compared with that of control (C, n = 12) individuals and determined the mechanism by which DM2-HSA can interfere in macrophage lipid accumulation. The HSA glycation level was analyzed by MALDI/MS. Macrophages were treated for 18 h with C- or DM2-HSA to measure the (14) C-cholesterol efflux, the intracellular lipid accumulation and the cellular ABCA-1 protein content. Agilent arrays (44000 probes) were used to analyze gene expression, and the differentially expressed genes were validated by real-time RT-PCR. An increased mean mass was observed in DM2-HSA compared with C-HSA, reflecting the condensation of at least 5 units of glucose. The cholesterol efflux mediated by apo AI, HDL3 , and HDL2 was impaired in DM2-HSA-treated cells, which was related to greater intracellular lipid accumulation. DM2-HSA decreased Abcg1 mRNA expression by 26%. Abca1 mRNA was unchanged, although the final ABCA-1 protein content decreased. Compared with C-HAS-treated cells, NADPH oxidase 4 mRNA expression increased in cells after DM2-HSA treatment. Stearoyl-Coenzyme A desaturase 1, janus kinase 2, and low density lipoprotein receptor mRNAs were reduced by DM2-HSA. The level of glycation that occurs in vivo in DM2-HSA-treated cells selectively alters macrophage gene expression, impairing cholesterol efflux and eliciting intracellular lipid accumulation, which contribute to atherogenesis, in individuals with DM2., (© 2014 Wiley Periodicals, Inc.)

Mesangial cells subject to high extracellular glucose concentrations, as occur in hyperglycaemic states, are unable to down regulate glucose influx, resulting in intracellular activation of deleterious biochemical pathways. A high expression of GLUT1 participates in the development of diabetic glomerulopathy. Variants in the gene encoding GLUT1 (SLC2A1) have been associated to this diabetic complication. The aim of this study was to test whether polymorphisms in SLC2A1 confer susceptibility to diabetic nephropathy (DN) in Brazilian type 1 diabetes patients. Four polymorphisms (rs3820589, rs1385129, rs841847 and rs841848) were genotyped in a Brazilian cohort comprised of 452 patients. A prospective analysis was performed in 155 patients. Mean duration of follow-up was 5.6 ± 2.4 years and the incidence of renal events was 18.0%. The rs3820589 presented an inverse association with the prevalence of incipient DN (OR: 0.36, 95% CI: 0.16 - 0.80, p=0.01) and with progression to renal events (HR: 0.20; 95% CI: 0.03 - 0.70; p=0.009). AGGT and AGAC haplotypes were associated with the prevalence of incipient DN and the AGAC haplotype was also associated with the prevalence of established/advanced DN. In conclusion, rs3820589 in the SLC2A1 gene modulates the risk to DN in Brazilian patients with inadequate type 1 diabetes control., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Decreased expression of glucose transporter protein GLUT4, encoded by the solute carrier 2A4 (Slc2a4) gene, is involved in obesity-induced insulin resistance. Local tissue inflammation, by nuclear factor-κB (NFκB)-mediated pathway, has been related to Slc2a4 repression; a mechanism that could be modulated by statins. Using a model of obesity with insulin resistance, this study investigated whether (1) inflammatory markers and Slc2a4 expression are altered; (2) atorvastatin has beneficial effects on inflammation and Slc2a4 expression; and (3) inhibitor of NFκB (IKK)/NFκB pathway is involved in subcutaneous adipose tissue (SAT)., Findings: Obese mice showed insulin resistance, decreased expression of Slc2a4 mRNA (66%, P < 0.01) and GLUT4 protein (30%, P < 0.05), and increased expression of interleukin 6 (Il6) mRNA (44%, P < 0.05) in SAT. Obese mice treated with atorvastatin had enhanced in vivo insulin sensitivity, besides increased Slc2a4/GLUT4 expression and reduced Il6 expression in SAT. No alterations of tumor necrosis factor-α, interleukin 1β and adiponectin expression or IKKα/β activity in SAT of obese mice or obese mice treated with atorvastatin were observed., Conclusions: Atorvastatin has beneficial effect upon glycemic homeostasis, which may be related to its positive impact on Il6 and Slc2a4/GLUT4 expression in SAT.

Oleic (OA) and linoleic (LA) fatty acids may be important regulators of Slc2a4 gene (GLUT4 protein) in skeletal muscle, thus participating in insulin resistance. We investigated the effect of OA and LA on the Slc2a4/GLUT4 expression in L6 muscle cells; as well as potential transcriptional regulators. OA and LA (50-400 µM) decreased the Slc2a4/GLUT4 expression in a dose-dependent way (maximum of ~50%, P < 0.001). OA and LA did not alter the Slc2a4-binding activity of oxysterols-receptor-LXR-alpha and peroxisome-proliferator-activated-receptor-gamma; but decreased the Slc2a4-binding activity of the sterol-regulatory-element-binding-protein-1 (SREBP1) enhancer (50%, P < 0.001), and increased (~30%, P < 0.001) the nuclear proteins binding into the Slc2a4-nuclear-factor-NF-kappa-B-binding site (repressor), and the phosphorylation of the inhibitors of nuclear-factor-kappa-B-kinase alpha/beta (150-300%, P < 0.001). In sum, OA and LA are potent inhibitors of the Slc2a4/GLUT4 expression in muscle cells; an effect involving reduced SREBP1 and increased NFKB transcriptional activity. These regulations may participate in the fatty acid-related pathophysiology of insulin resistance., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

We characterized the metabolic profile of transgenic mice exhibiting enhanced muscle mass driven by increased mIGF-1 expression (MLC/mIGF-1). As expected, 6-month-old MLC/mIGF-1 mice were heavier than age-matched wild type (WT) mice (37.4 ± 0.3 versus 31.8 ± 0.6 g, resp.). MLC/mIGF-1 mice had higher respiratory quotient when compared to WT (0.9 ± 0.03 versus 0.74 ± 0.02, resp.) suggesting a preference for carbohydrate as the major fuel source. MLC/mIGF-1 mice had a higher rate of glucose disposal when compared to WT (3.25 ± 0.14 versus 2.39 ± 0.03%/min, resp.). The higher disposal rate correlated to ∼ 2-fold higher GLUT4 content in the extensor digitorum longus (EDL) muscle. Analysis of mRNA content for the glycolysis-related gene PFK-1 showed ∼ 3-fold upregulation in MLC/mIGF-1 animals. We also found a 50% downregulation of PGC1α mRNA levels in MLC/mIGF-1 mouse EDL muscle, suggesting less abundant mitochondria in this tissue. We found no difference in the expression of PPARα and PPARβ/δ, suggesting no modulation of key elements in oxidative metabolism. These data together suggest a shift in metabolism towards higher carbohydrate utilization, and that could explain the increased insulin sensitivity of hypertrophied skeletal muscle in MLC/mIGF-1 mice.

Statins have a beneficial effect after myocardial infarction, but the relationship between glucose transporters and their use before the event has not yet been studied. We assessed the effects of atorvastatin treatment pre- and post-myocardial infarction on cardiovascular function and glucose transporter 4 (GLUT4) in the heart. Wistar-Kyoto rats were treated with 20 mg/kg atorvastatin or vehicle for 14 days before coronary artery occlusion surgery (myocardial infarction) or sham surgery. Echocardiographic evaluations were carried out 48 h after myocardial infarction (protocol A) and after 7 days (protocol B), when atorvastatin was also administered. Plasma inflammatory markers and GLUT4 in the heart were also evaluated. Animals were divided into the following groups: sham-operated and vehicle (C), myocardial infarction and vehicle (I), sham-operated and atorvastatin (CAt) and myocardial infarction and atorvastatin (IAt). After 48 h, myocardial infarction induced higher left ventricular fractional shortening in IAt versus I (~ 60%, P = 0.036), and the ejection fraction was lower (protocol A ~ 37%; protocol B ~ 30%). Myocardial infarction was associated with a rise in plasma membrane GLUT4 after 48 h (~ 40%, P < 0.001), and a reduction in GLUT4 after 7 days (I 25%; IAt 49%, P < 0.001). Atorvastatin treatment for 48 h after the infarction did not change GLUT4 expression, and after 7 days it had an additional negative effect on GLUT4 content (~ 39%, P = 0.030). In conclusion, atorvastatin treatment pre- and post-myocardial infarction improved myocardial contractility after 48 h, but not after 7 days, and was not associated with an increase in GLUT4 expression., (© 2014 Wiley Publishing Asia Pty Ltd.)

Aims: The insulin-sensitive glucose transporter protein GLUT4 (solute carrier family 2 member 4 (Slc2a4) gene) plays a key role in glycemic homeostasis. Decreased GLUT4 expression is a current feature in insulin resistant conditions such as diabetes, and the restoration of GLUT4 content improves glycemic control. This study investigated the effect of insulin upon Slc2a4/GLUT4 expression, focusing on the AT-rich element, E-box and nuclear factor NF-kappa-B (NFKB) site., Main Methods: Rat soleus muscles were incubated during 180 min with insulin, added or not with wortmannin (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (PI3K)-inhibitor), ML9 (serine/threonine protein kinase (AKT) inhibitor) and tumor necrosis factor (TNF, GLUT4 repressor), and processed for analysis of GLUT4 protein (Western blotting); Slc2a4, myocyte enhancer factor 2a/d (Mef2a/d), hypoxia inducible factor 1a (Hif1a), myogenic differentiation 1 (Myod1) and nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (Nfkb1) messenger ribonucleic acids (mRNAs) (polymerase chain reaction (PCR)); and AT-rich- (myocyte-specific enhancer factor 2 (MEF2)-binding site), E-box- (hypoxia inducible factor 1 alpha (HIF1A)- and myoblast determination protein 1 (MYOD1)-binding site), and NFKB-binding activity (electrophoretic mobility assay)., Key Findings: Insulin increased Slc2a4 mRNA expression (140%) and nuclear proteins binding to AT-rich and E-box elements (~90%), all effects were prevented by wortmannin and ML9. Insulin also increased Mef2a/d and Myod1 mRNA expression, suggesting the participation of these transcriptional factors in the Slc2a4 enhancing effect. Conversely, insulin decreased Nfkb1 mRNA expression and protein binding to the NFKB-site (~50%). Furthermore, TNF-induced inhibition of GLUT4 expression (~40%) was prevented by insulin in an NFKB-binding repressing mechanism. GLUT4 protein paralleled the Slc2a4 mRNA regulations., Significance: Insulin enhances the Slc2a4/GLUT4 expression in the skeletal muscle by activating AT-rich and E-box elements, in a PI3K/AKT-dependent mechanism, and repressing NFKB-site activity as well. These results unravel how post-prandial increase of insulin may guarantee GLUT4 expression, and how the insulin signaling impairment can participate in insulin resistance-induced repression of GLUT4., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Aim: Investigate, in healthy sedentary rats, the potential mechanisms involved on the effects of beta hydroxy beta methylbutyrate (HMB) supplementation upon the glycaemic homeostasis, by evaluating the insulin sensitivity in liver, skeletal muscle, and white adipose tissue., Methods: Rats were supplemented with either beta hydroxy beta methylbutyrate (320 mg kg(-1)  BW) or saline by gavage for 4 weeks. After the experimental period, the animals were subjected to the glucose tolerance test (GTT) and plasma non-esterified fatty acids (NEFA) concentration measurements. The soleus skeletal muscle, liver and white adipose tissue were removed for molecular (western blotting and RT-PCR) and histological analysis., Results: The beta hydroxy beta methylbutyrate supplemented rats presented: (i) higher ratio between the area under the curve (AUC) of insulinaemia and glycaemia during glucose tolerance test; (ii) impairment of insulin sensitivity on liver and soleus skeletal muscle after insulin overload; (iii) reduction of glucose transporter 4 (GLUT 4) total and plasma membrane content on soleus; (iv) increased hormone-sensitive lipase (HSL) mRNA and protein expression on white adipose tissue and plasma NEFA levels and (v) reduction of fibre cross-sectional area of soleus muscle., Conclusion: The data altogether indicate that beta hydroxy beta methylbutyrate supplementation impairs insulin sensitivity in healthy sedentary rats, which, in the long-term, could lead to an increased risk of developing type 2 diabetes., (© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Objective: GLUT4 protein, encoded by the Slc2a4 gene, plays a key role in muscle glucose uptake, and its expression decreases in muscles under insulin resistance. Slc2a4/GLUT4 decreases with fasting and rapidly increases with refeeding and the same occurs to plasma glucose, amino acids, insulin and T3. Thus, they might be potential regulators of the Slc2a4 gene, which makes them promising targets for strategies to improve GLUT4 expression. Herein, we investigate the role of metabolic-hormonal parameters triggered by refeeding upon the Slc2a4 expression., Materials/methods: Plasma glucose/insulin/T3, and gastrocnemius Slc2a4 mRNA contents were measured in rats studied at the end of 48-h fasting, and subsequently at: i) 2-4h after spontaneous refeeding; ii) 2-4h after T3 injection, without refeeding; and iii) 0.5-2h after intravenous infusion of insulin, insulin+glucose and insulin+amino acids, without refeeding., Results: Refeeding increased plasma glucose/insulin/T3 and muscle Slc2a4 mRNA, reverting insulin resistance. Post-fasting infusions surprisingly induced a further Slc2a4 mRNA decrease (~20%, P<0.05 vs. fasting), but T3 injection induced a ~2-fold increase in Slc2a4 mRNA, 2-4h later (P<0.001). Moreover, T3 increased glycemia and insulinemia to the 2h-refed rats levels, suggesting that T3 elevation is a key factor to the mechanisms of metabolic balance during refeeding., Conclusions: Refeeding induces a rapid increase in muscle Slc2a4 expression, not associated with increased plasma glucose, insulin or amino acids, but highly correlated to increased plasma T3 concentration. This result points out T3 hormone as a powerful Slc2a4 enhancer, an effect that may be acutely explored in situations of insulin resistance., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Aims: In models of diabetes, distal nephron cells contribute to glucose uptake and oxidation. How these cells contribute to the use of glucose for the regulation of H(+) extrusion remains unknown. We used Madin-Darby Canine Kidney (MDCK) cells to investigate the effect of acute or chronic high glucose concentration on the abundance and activity of the Na(+)/H(+) exchanger (NHE-1)., Methods: Using RT-PCR, we also evaluated the mRNA expression for sodium glucose co-transporters SGLT1 and SGLT2. Protein abundance was analyzed using immunoblotting, and intracellular pH (pHi) recovery was evaluated using microscopy in conjunction with the fluorescent probe BCECF/AM. The Na(+)-dependent pHi recovery rate was monitored with HOE-694 (50 µM) and/or S3226 (10 µM), specific NHE-1 and NHE-3 inhibitors., Results: MDCK cells did not express the mRNA for SGLT1 or SGLT2 but did express the GLUT2, NHE-1 and NHE-3 proteins. Under control conditions, we observed a greater contribution of NHE-1 to pHi recovery relative to the other H(+) transporters. Acute high glucose treatment increased the HOE-694-sensitive pHi recovery rate and p-Erk1/2 and p90(RSK) abundance. These parameters were reduced by PD-98059, a Mek inhibitor (1 µM). Chronic high glucose treatment also increased the HOE-694-sensitive pHi recovery rate and p-p38MAPK abundance. Both parameters were reduced by SB-203580, a p38MAPK inhibitor (10 µM)., Conclusion: These results suggested that extracellular high glucose stimulated NHE-1 acutely and chronically through Mek/Erk1/2/p90(RSK) and p38MAPK pathways, respectively., (© 2014 S. Karger AG, Basel.)

The canine corpus luteum (CL) functions as a source of progesterone (P4) and 17β-oestradiol (E2); however, the transport of energy substrates to maintain its high hormonal output has not yet been characterised. This study involved the localisation and temporal distribution of the facilitative glucose transporter 1 and the quantification of the corresponding protein (GLUT1) and gene (SLC2A1) expression. Some GLUT1/SLC2A1 regulatory proteins, such as hypoxia-inducible factor 1α (HIF1A) and fibroblast growth factor 2 (FGF2); mRNAs, such as HIF1A, FGF2 and vascular endothelial growth factor A (VEGFA); and VEGFA receptors 1 and 2 (FLT1 and KDR) were also analysed from days 10 to 70 after ovulation. Additionally, plasma P4 and E2 levels were assessed via chemiluminescence. Moreover, the canine KDR sequence has been cloned, thereby enabling subsequent semi-quantitative PCR analysis. Our results demonstrate time-dependent variations in the expression profile of SLC2A1 during dioestrus, which were accompanied by highly correlated changes (0.84

Aims: Solute carrier 2a2 (Slc2a2) gene codifies the glucose transporter GLUT2, a key protein for glucose flux in hepatocytes and renal epithelial cells of proximal tubule. In diabetes mellitus, hepatic and tubular glucose output has been related to Slc2a2/GLUT2 overexpression; and controlling the expression of this gene may be an important adjuvant way to improve glycemic homeostasis. Thus, the present study investigated transcriptional mechanisms involved in the diabetes-induced overexpression of the Slc2a2 gene., Main Methods: Hepatocyte nuclear factors 1α and 4α (HNF-1α and HNF-4α), forkhead box A2 (FOXA2), sterol regulatory element binding protein-1c (SREBP-1c) and the CCAAT-enhancer-binding protein (C/EBPβ) mRNA expression (RT-PCR) and binding activity into the Slc2a2 promoter (electrophoretic mobility assay) were analyzed in the liver and kidney of diabetic and 6-day insulin-treated diabetic rats., Key Findings: Slc2a2/GLUT2 expression increased by more than 50% (P<0.001) in the liver and kidney of diabetic rats, and 6-day insulin treatment restores these values to those observed in non-diabetic animals. Similarly, the mRNA expression and the binding activity of HNF-1α, HNF-4α and FOXA2 increased by 50 to 100% (P<0.05 to P<0.001), also returning to values of non-diabetic rats after insulin treatment. Neither the Srebf1 and Cebpb mRNA expression, nor the SREBP-1c and C/EBP-β binding activity was altered in diabetic rats., Significance: HNF-1α, HNF-4α and FOXA2 transcriptional factors are involved in diabetes-induced overexpression of Slc2a2 gene in the liver and kidney. These data point out that these transcriptional factors are important targets to control GLUT2 expression in these tissues, which can contribute to glycemic homeostasis in diabetes., (© 2013.)

Background: Oral health complications in diabetes and hypertension include decreased salivary secretion. The sodium-glucose cotransporter 1 (SGLT1) protein, which transports 1 glucose/2 Na+/264 H2O molecules, is described in salivary glands. We hypothesized that changes in SGLT1 expression in the luminal membrane of ductal cell may be related to an altered salivary flow., Findings: By immunohistochemistry, we investigated SGLT1 expression in ductal cells of parotid and submandibular glands from Wistar Kyoto rats (WKY), diabetic WKY (WKY-D), spontaneously hypertensive rats (SHR) and diabetic SHR (SHR-D), as well as in parotid glands from WKY subjected to sympathetic stimulation, with or without previous propranolol blockade. Diabetes and hypertension decreased the salivary secretion and increased SGLT1 expression in the luminal membrane of ductal cells, and their association exacerbated the regulations observed. After 30 min of sympathetic stimulation, SGLT1 increased in the luminal membrane of ductal cells, and that was blocked by previous injection of propranolol., Conclusions: SGLT1 expression increases in the luminal membrane of salivary gland ductal cells and the salivary flow decreases in diabetic and hypertensive rats, which may be related to sympathetic activity. This study highlights the water transporter role of SGLT1 in salivary glands, which, by increasing ductal water reabsorption, may explain the hyposalivation of diabetic and hypertensive subjects.

Considering the similarity between structural, hemodynamic, and functional changes of obesity-related renal disease and diabetic nephropathy, we hypothesized that renal glucose transporter changes occur in obesity as in diabetes. The aim of the work was to evaluate GLUT1 and GLUT2 in kidneys of an animal model of metabolic syndrome. Neonate spontaneously hypertensive rats (SHR), n=15/group, were treated with monosodium glutamate (5 mg/g) (MetS) for 9 days and compared with saline-treated Wistar-Kyoto (C) and SHR (H) rats. Lee index, systolic arterial pressure (SAP), glycemia, insulin resistance, triglycerides, and HDL cholesterol were evaluated at 3 and 6 months. Medullar GLUT1 and cortical GLUT2 were analyzed by Western blot. MetS vs. C and H rats had the highest Lee index (p<0.001) and insulin resistance (3-months C: 4.3±0.7, H: 3.9±0.9, MetS: 2.7±0.6; 6-months C: 4.2±0.6, H: 3.8±0.5, MetS: 2.4±0.6% · min⁻¹, p<0.001), similar glycemia, and the lowest HDL-cholesterol at 6-months (p<0.001). In the MetS and H rats, SAP was higher vs. C at 3-months (p<0.001) and 6-months (C: 151±15, H: 190±11, MetS: 185±13 mm Hg, p<0.001) of age. GLUT1 was ̴ 13× lower (p<0.001) at 3-months, reestablishing its content at 6-months in MetS group, while GLUT2 was 2× higher (p<0.001) in this group at 6-months of age. Renal GLUT1 and GLUT2 are modulated in kidney of rats with metabolic syndrome, where obesity, insulin resistance and hypertension coexist, despite normoglycemia. Like in diabetes, cortical GLUT2 overexpression may contribute to the development of kidney disease., (© Georg Thieme Verlag KG Stuttgart · New York.)

Aims: Angiotensin-converting enzyme (ACE) inhibitors are used in diabetic kidney disease to reduce systemic/intra-glomerular pressure. The objective of this study was to investigate whether reducing blood pressure (BP) could modulate renal glucose transporter expression, and urinary markers of diabetic nephropathy in diabetic hypertensive rats treated with ramipril or amlodipine., Main Methods: Diabetes was induced in spontaneously-hypertensive rats (~210 g) by streptozotocin (50mg/kg). Thirty days later, animals received ramipril 15 μg/kg/day (R, n=10), or amlodipine 10mg/kg/day (A, n=8,) or water (C, n=10) by gavage. After 30-day treatment, body weight, glycaemia, urinary albumin and TGF-β1 (enzyme-linked immunosorbent assay) and BP (tail-cuff pressure method) were evaluated. Kidneys were removed for evaluation of renal cortex glucose transporters (Western blotting) and renal tissue ACE activity (fluorometric assay)., Key Findings: After treatments, body weight (p=0.77) and glycaemia (p=0.22) were similar among the groups. Systolic BP was similarly reduced (p<0.001) in A and R vs. C (172.4 ± 3.2; 1867 ± 3.7 and 202.2 ± 4.3 mmHg; respectively). ACE activity (C: 0.903 ± 0.086; A: 0.654 ± 0.025, and R: 0.389 ± 0.057 mU/mg), albuminuria (C: 264.8 ± 15.4; A: 140.8 ± 13.5 and R: 102.8 ± 6.7 mg/24h), and renal cortex GLUT1 content (C: 46.81 ± 4.54; A: 40.30 ± 5.39 and R: 26.89 ± 0.79 AU) decreased only in R (p<0.001, p<0.05 and p<0.001; respectively)., Significance: We concluded that the blockade of the renin-angiotensin system with ramipril reduced early markers of diabetic nephropathy, a phenomenon that cannot be specifically related to decreased BP levels., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objectives: We evaluated the effects of aerobic exercise training without dietary changes on cardiovascular and metabolic variables and on the expression of glucose transporter Type 4 in rats with metabolic syndrome., Methods: Twenty male spontaneously hypertensive rats received monosodium glutamate during the neonatal period. The animals were allocated to the following groups: MS (sedentary metabolic syndrome), MS-T (trained on a treadmill for 1 hour/day, 5 days/week for 10 weeks), H (sedentary spontaneously hypertensive rats) and H-T (trained spontaneously hypertensive rats). The Lee index, blood pressure (tail-cuff system), insulin sensitivity (insulin tolerance test) and functional capacity were evaluated before and after 10 weeks of training. Glucose transporter Type 4 expression was analyzed using Western blotting. The data were compared using analysis of variance (ANOVA) (p<0.05)., Results: At baseline, the MS rats exhibited lower insulin sensitivity and increased Lee index compared with the H rats. Training decreased the body weight and Lee index of the MS rats (MS-T vs. MS), but not of the H rats (H-T vs. H). There were no differences in food intake between the groups. At the end of the experiments, the systolic blood pressure was lower in the two trained groups than in their sedentary controls. Whole-body insulin sensitivity increased in the trained groups. Glucose transporter Type 4 content increased in the heart, white adipose tissue and gastrocnemius muscle of the trained groups relative to their respective untrained groups., Conclusion: In conclusion, the present study shows that an isolated aerobic exercise training intervention is an efficient means of improving several components of metabolic syndrome, that is, training reduces obesity and hypertension and increases insulin sensitivity.

Objective: Validate a model of high-fat diet-induced obesity, of low cost, easy reproducibility, that could express characteristics observed in human, and would enable subsequent therapy proposals., Materials and Methods: Sixteen Swiss mice received a standard diet (DP) or high-fat diet (DH) for 10 weeks., Results: Although the DP group had greater water (p < 0.01) and feed (p < 0.001) consumption, the DH group had greater body weight (p < 0.5) and adipose tissue gain (p < 0.001), favoring higher adiposity index (p < 0.001), glucose (p < 0.01), and area under the curve in the insulin (p < 0.001) and glucose (p < 0.01) tolerance tests., Conclusion: A high-fat diet-induced obesity model has been validated, which was also associated with insulin resistance and glucose intolerance after a period of 10 weeks.

Glucose transporter GLUT4 protein, codified by Slc2a4 gene plays a key role in glycemic homeostasis. Insulin resistance, as in obesity, has been associated to inflammatory state, in which decreased GLUT4 is a feature. Inflammatory NF-κB transcriptional factor has been proposed as a repressor of Slc2a4; although, the binding site(s) in Slc2a4 promoter and the direct repressor effect have never been reported yet. A motif-based sequence analysis of mouse Slc2a4 promoter revealed two putative κB sites located inside -83/-62 and -134/-113 bp. Eletrophoretic mobility assay showed that p50 and p65 NF-κB subunits bind to both putative κB sites. Chromatin immunoprecipitation assay using genomic DNA from adipocytes confirmed p50- and p65-binding to Slc2a4 promoter. Moreover, transfection experiments revealed that NF-κB binds to the -134/-113bp region of the mouse Slc2a4 gene promoter, inhibiting the Slc2a4 gene transcription. The current findings demonstrate the existence of two κB sites in Slc2a4 gene promote, and that NF-κB has a direct repressor effect upon the Slc2a4 gene, providing an important link between insulin resistance and inflammation., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Interleukin-6 (IL-6) has a dual role in modulating insulin sensitivity, with evidence for this cytokine as both an enhancer and inhibitor of insulin action. We determined the effect of IL-6 exposure on glucose and lipid metabolism in cultured myotubes established from people with normal glucose tolerance or type 2 diabetes. Acute IL-6 exposure increased glycogen synthesis, glucose uptake, and signal transducer and activator of transcription 3 (STAT3) phosphorylation in cultured myotubes from normal glucose tolerant subjects. However, in type 2 diabetic patients, IL-6 was without effect on glucose metabolism and STAT3 signaling, concomitant with increased suppressor of cytokine signaling 3 (SOCS3) expression. IL-6 increased fatty acid oxidation in myotubes from type 2 diabetic and normal glucose tolerant subjects. Expression of IL-6, IL-6 receptor (IL-6R), or glycoprotein 130, as well as IL-6 secretion, was unaltered between cultured myotubes from normal glucose tolerant or type 2 diabetic subjects. Circulating serum IL-6 concentration was unaltered between normal glucose tolerant and type 2 diabetic subjects. In summary, skeletal muscle cells from type 2 diabetic patients display selective IL-6 resistance for glucose rather than lipid metabolism. In conclusion, IL-6 appears to play a differential role in regulating metabolism in type 2 diabetic patients compared with normal glucose tolerant subjects.

Objective: To evaluate the effect of exposure to cigarette smoke and running training on a treadmill on the expression of glucose transporter GLUT4 in oxidative soleus muscle of rats., Materials and Methods: Wistar rats were divided into: (C) control, (E) exercise control, (SS), sedentary smoker, and (ES) exercise smoker. Insulin Tolerance Test, Western blotting, and RT-PCR were performed for the evaluation of GLUT4 levels., Results: The SS group presented lower insulin sensitivity with reduced GLUT4 protein in the plasma membrane (PM), no changes in the microsomal fraction, but increased mRNA content. Training reversed this condition. No intervention altered total GLUT4 content of the oxidative muscle., Conclusion: These results suggest that passive smoking stimulates GLUT4 transcription without changing total protein content, but impairs the ability of GLUT4 translocation to the PM. On the other hand, training seems to reduce the deleterious effects, even under the influence of cigarette smoking.

Insulin and the inhibition of the renin-angiotensin system have independent benefits for ischemia-reperfusion injury, but their combination has not been tested. Our aim was to evaluate the effects of insulin+captopril on insulin/angiotensin signaling pathways and cardiac function in the isolated heart subjected to ischemia-reperfusion. Isolated hearts were perfused (Langendorff technique) with Krebs-Henseleit (KH) buffer for 25 min. Global ischemia was induced (20 min), followed by reperfusion (30 min) with KH (group KH), KH+angiotensin-I (group A), KH+angiotensin-I+captopril (group AC), KH+insulin (group I), KH+insulin+angiotensin-I (group IA), or KH+insulin+angiotensin-I+captopril (group IAC). Group A had a 24% reduction in developed pressure and an increase in end-diastolic pressure vs. baseline, effects that were reverted in groups AC, IA, and IAC. The phosphorylation of protein kinase B (AKT) was higher in groups I and IA vs. groups KH and A. The phosphorylation of AMP-activated protein kinase (AMPK) was ∼31% higher in groups I, IA, and IAC vs. groups KH, A, and AC. The tert-butyl hydroperoxide (tBOOH)-induced chemiluminescence was lower (∼2.2 times) in all groups vs. group KH and was ∼35% lower in group IA vs. group A. Superoxide dismutase content was lower in groups A, AC, and IAC vs. group KH. Catalase activity was ∼28% lower in all groups (except group IA) vs. group KH. During reperfusion of the ischemic heart, insulin activates the AKT and AMPK pathways and inhibits the deleterious effects of angiotensin-I perfusion on SOD expression and cardiac function. The addition of captopril does not potentiate these effects., (© 2011 The Authors Fundamental and Clinical Pharmacology © 2011 Société Française de Pharmacologie et de Thérapeutique.)

Salivary gland function is regulated by both the sympathetic and parasympathetic nervous systems. Previously we showed that the basal sympathetic outflow to the salivary glands (SNA(SG)) was higher in hypertensive compared to normotensive rats and that diabetes reduced SNA(SG) discharge at both strains. In the present study we sought to investigate how SNA(SG) might be modulated by acute changes in the arterial pressure and whether baroreceptors play a functional role upon this modulation. To this end, we measured blood pressure and SNA(SG) discharge in Wistar-Kyoto rats (WKY-intact) and in WKY submitted to sinoaortic denervation (WKY-SAD). We made the following three major observations: (i) in WKY-intact rats, baroreceptor loading in response to intravenous infusion of the phenylephrine evoked an increase in SNA(SG) spike frequency (81%, p<0.01) accompanying the increase mean arterial pressure (ΔMAP: +77 ± 14 mmHg); (ii) baroreceptor unloading with sodium nitroprusside infusion elicited a decrease in SNA(SG) spike frequency (17%, p<0.01) in parallel with the fall in arterial blood pressure (ΔMAP: -30 ± 3 mmHg) in WKY-intact rats; iii) in the WKY-SAD rats, phenylephrine-evoked rises in the arterial pressure (ΔMAP: +56 ± 6 mmHg) failed to produce significant changes in the SNA(SG) spike frequency. Taken together, these data show that SNA(SG) increases in parallel with pharmacological-induced pressor response in a baroreceptor dependent way in anaesthetised rats. Considering the key role of SNA(SG) in salivary secretion, this mechanism, which differs from the classic cardiac baroreflex feedback loop, strongly suggests that baroreceptor signalling plays a decisive role in the regulation of salivary gland function., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objectives: The aim of this study was to evaluate cardiovascular autonomic function in a rodent obesity model induced by monosodium glutamate injections during the first seven days of life., Method: The animals were assigned to control (control, n = 10) and monosodium glutamate (monosodium glutamate, n = 13) groups. Thirty-three weeks after birth, arterial and venous catheters were implanted for arterial pressure measurements, drug administration, and blood sampling. Baroreflex sensitivity was evaluated according to the tachycardic and bradycardic responses induced by sodium nitroprusside and phenylephrine infusion, respectively. Sympathetic and vagal effects were determined by administering methylatropine and propranolol., Results: Body weight, Lee index, and epididymal white adipose tissue values were higher in the monosodium glutamate group in comparison to the control group. The monosodium glutamate-treated rats displayed insulin resistance, as shown by a reduced glucose/insulin index (-62.5%), an increased area under the curve of total insulin secretion during glucose overload (39.3%), and basal hyperinsulinemia. The mean arterial pressure values were higher in the monosodium glutamate rats, whereas heart rate variability (>7 times), bradycardic responses (>4 times), and vagal (~38%) and sympathetic effects (~36%) were reduced as compared to the control group., Conclusion: Our results suggest that obesity induced by neonatal monosodium glutamate treatment impairs cardiac autonomic function and most likely contributes to increased arterial pressure and insulin resistance.

Background: Metabolic syndrome is characterized by insulin resistance, which is closely related to GLUT4 content in insulin-sensitive tissues. Thus, we evaluated the GLUT4 expression, insulin resistance and inflammation, characteristics of the metabolic syndrome, in an experimental model., Methods: Spontaneously hypertensive neonate rats (18/group) were treated with monosodium glutamate (MetS) during 9 days, and compared with Wistar-Kyoto (C) and saline-treated SHR (H). Blood pressure (BP) and lipid levels, C-reactive protein (CRP), interleukin 6 (IL-6), TNF-α and adiponectin were evaluated. GLUT4 protein was analysed in the heart, white adipose tissue and gastrocnemius. Studies were performed at 3 (3-mo), 6 (6-mo) and 9 (9-mo) months of age., Results: MetS rats were more insulin resistant (p<0.001, all ages) and had higher BP (3-mo: p<0.001, 6-mo: p = 0.001, 9-mo: p = 0.015) as compared to C. At 6 months, CRP, IL-6 and TNF-α were higher (p<0.001, all comparisons) in MetS rats vs H, but adiponectin was lower in MetS at 9 months (MetS: 32 ± 2, H: 42 ± 2, C: 45 ± 2 pg/mL; p<0.001). GLUT4 protein was reduced in MetS as compared to C rats at 3, 6 and 9-mo, respectively (Heart: 54%, 50% and 57%; Gastrocnemius: 37%, 56% and 50%; Adipose tissue: 69%, 61% and 69%)., Conclusions: MSG-treated SHR presented all metabolic syndrome characteristics, as well as reduced GLUT4 content, which must play a key role in the impaired glycemic homeostasis of the metabolic syndrome.

Quercetin is a potent anti-inflammatory flavonoid, but its capacity to modulate insulin sensitivity in obese insulin resistant conditions is unknown. This study investigated the effect of quercetin treatment upon insulin sensitivity of ob/ob mice and its potential molecular mechanisms. Obese ob/ob mice were treated with quercetin for 10 weeks, and L6 myotubes were treated with either palmitate or tumor necrosis factor-α (TNFα) plus quercetin. Cells and muscles were processed for analysis of glucose transporter 4 (GLUT4), TNFα and inducible nitric oxide synthase (iNOS) expression, and c-Jun N-terminal kinase (JNK) and inhibitor of nuclear factor-κB (NF-κB) kinase (IκK) phosphorylation. Myotubes were assayed for glucose uptake and NF-κB translocation. Chromatin immunoprecipitation assessed NF-κB binding to GLUT4 promoter. Quercetin treatment improved whole body insulin sensitivity by increasing GLUT4 expression and decreasing JNK phosphorylation, and TNFα and iNOS expression in skeletal muscle. Quercetin suppressed palmitate-induced upregulation of TNFα and iNOS and restored normal levels of GLUT4 in myotubes. In parallel, quercetin suppressed TNFα-induced reduction of glucose uptake in myotubes. Nuclear accumulation of NF-κB in myotubes and binding of NF-κB to GLUT4 promoter in muscles of ob/ob mice were also reduced by quercetin. We demonstrated that quercetin decreased the inflammatory status in skeletal muscle of obese mice and in L6 myotubes. This effect was followed by increased muscle GLUT4, with parallel improvement of insulin sensitivity. These results point out quercetin as a putative strategy to manage inflammatory-related insulin resistance., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Evidences have suggested that the endocannabinoid system is overactive in obesity, resulting in enhanced endocannabinoid levels in both circulation and visceral adipose tissue. The blockade of cannabinoid receptor type 1 (CB1) has been proposed for the treatment of obesity. Besides loss of body weight, CB1 antagonism improves insulin sensitivity, in which the glucose transporter type 4 (GLUT4) plays a key role. The aim of this study was to investigate the modulation of GLUT4-encoded gene (Slc2a4 gene) expression by CB1 receptor. For this, 3T3-L1 adipocytes were incubated in the presence of a highly selective CB1 receptor agonist (1 μM arachidonyl-2'-chloroethylamide) and/or a CB1 receptor antagonist/inverse agonist (0.1, 0.5, or 1 μM AM251, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide). After acute (2 and 4 h) and chronic (24 h) treatments, cells were harvested to evaluate: i) Slc2a4, Cnr1 (CB1 receptor-encoded gene), and Srebf1 type a (SREBP-1a type-encoded gene) mRNAs (real-time PCR); ii) GLUT4 protein (western blotting); and iii) binding activity of nuclear factor (NF)-κB and sterol regulatory element-binding protein (SREBP)-1 specifically in the promoter of Slc2a4 gene (electrophoretic mobility shift assay). Results revealed that both acute and chronic CB1 receptor antagonism greatly increased (∼2.5-fold) Slc2a4 mRNA and protein content. Additionally, CB1-induced upregulation of Slc2a4 was accompanied by decreased binding activity of NF-κB at 2 and 24 h, and by increased binding activity of the SREBP-1 at 24 h. In conclusion, these findings reveal that the blockade of CB1 receptor markedly increases Slc2a4/GLUT4 expression in adipocytes, a feature that involves NF-κB and SREBP-1 transcriptional regulation.

Background: The purpose of this study is to investigate whether local inflammatory events, such as periodontal disease, are able to increase tumor necrosis factor-alpha (TNF-α) plasmatic concentration and decrease insulin sensitivity and insulin signaling in non-diabetic rats., Methods: Forty-eight male Wistar rats (2 months old) were divided into two groups, with either ligature-induced periodontal disease (LPD) or control conditions (CN). Experiments were performed in both groups 28 days after ligature placement. Plasmatic concentration of glycemia and TNF-α (n = 10) were analyzed by the glucose oxidase and enzyme-linked immunosorbent assay method, respectively. Insulin sensitivity (n = 7) was measured using the insulin tolerance test. Insulin signal transduction (n = 7) was measured by pp185 tyrosine phosphorylation status in insulin-sensitive tissues using the Western blotting method., Results: The LPD group showed decreased insulin sensitivity (P <0.05), although no glycemic alterations were noted (P >0.05). TNF-α plasmatic concentration was higher in LPD rats compared to CN rats. In addition, a decrease in the pp185 tyrosine phosphorylation status was observed after insulin stimulus in both white adipose and skeletal muscle tissues of the LPD group compared with the CN group., Conclusions: LPD is able to cause alterations to both insulin signaling and insulin sensitivity, probably because of the elevation of TNF-α plasmatic concentration. Thus, the present results emphasize the importance of the prevention of local inflammatory diseases, such as periodontitis, to prevent diabetes mellitus.

Chronic intake of high-carbohydrate or high-lipid diets is a well-known insulin resistance inducer. This study investigates the immediate effect (1-6 h) of a carbohydrate- or lipid-enriched meal on insulin sensitivity. Fasted rats were refed with standard, carbohydrate-enriched (C), or lipid-enriched (L) meal. Plasma insulin, glucose, and non-esterified fatty acids (NEFA) were measured at 1, 2, 4, and 6 h of refeeding. The glucose-insulin index showed that either carbohydrates or lipids decreased insulin sensitivity at 2 h of refeeding. At this time point, insulin tolerance tests (ITTs) and glucose tolerance tests (GTTs) detected insulin resistance in C rats, while GTT confirmed it in L rats. Reduced glycogen and phosphorylated AKT and GSK3 content revealed hepatic insulin resistance in C rats. Reduced glucose uptake in skeletal muscle subjected to the fatty acid concentration that mimics the high NEFA level of L rats suggests insulin resistance in these animals is mainly in muscle. In conclusion, carbohydrate- or lipid-enriched meals acutely disrupt glycemic homeostasis, inducing a transient insulin resistance, which seems to involve liver and skeletal muscle, respectively. Thus, the insulin resistance observed when those types of diets are chronically consumed may be an evolution of repeated episodes of this transient insulin resistance.

Type 2 diabetes mellitus is characterized by disruption in glycemic homeostasis, involving impaired insulin-induced glucose disposal. For that, reduced glucose transporter GLUT4, encoded by Slc2a4 gene, plays a fundamental role. Conversely, increase in Slc2a4/GLUT4 expression improves glycemic homeostasis. Recent studies have proposed that estradiol is able to modulate Slc2a4 expression, according to distinct effects upon estrogen receptors ESR1/ESR2. We hypothesize that ESR1-agonist effect could stimulate Slc2a4 expression; thus, increasing cellular glucose disposal, which could be beneficial to glycemic control. Differentiated 3T3-L1 adipocytes were treated (24 hours) with selective ESR1- agonist PPT 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole, selective ESR1-antagonist MPP 1,3-Bis(4- hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride, and selective ESR2 agonist DPN 2,3-bis(4-Hydroxyphenyl)-propionitrile, with/without 17β-estradiol (E2). We analyzed Slc2a4 mRNA (real time PCR) and GLUT4 protein (Western blotting) expression, transcriptional activity of the Slc2a4 repressor Nuclear Factor- κB (NF-κB) (electrophoretic mobility shift assay), and cellular glucose disposal (2-deoxi-D-[(3)H]glucose uptake, 2-DG). ESR1-agonist PPT enhanced Slc2a4/GLUT4 expression (~30%) in the absence or presence of 0.1 and 10 nmol/L E2, and decreased the NF-κB binding activity (~50%). Conversely, ESR1-antagonist MPP, together with E2, decreased Slc2a4/GLUT4 expression (20-40%) and increased NF-κB binding activity (~30%). Furthermore, treatment with ESR2- agonist DPN decreased Slc2a4/GLUT4 expression (20-50%). 2-DG uptake was modulated in parallel to that observed in GLUT4 protein. The present results reveal that ESR1 activity enhances, whereas ESR2 activity represses, Slc2a4/GLUT4 expression. These effects are partially mediated by NF-κB, and allow parallel changes in adipocyte glucose disposal. Furthermore, the data provide evidences that ESR1-agonist PPT, as a Slc2a4/GLUT4 enhancer, can be a promising coadjuvant drug for diabetes mellitus therapy.

Background: Glucose transporter 4 (GLUT4) is highly expressed in muscle and fat tissue, where triiodothyronine (T(3)) induces solute carrier family 2 facilitated glucose transporter member 4 (SLC2A4) gene transcription. T(3) was also shown to rapidly increase glucose uptake in myocytes exposed to cycloheximide, indicating that it might act nongenomically to regulate GLUT4 availability. We tested this hypothesis by evaluating, in thyroidectomized rats (Tx rats), the acute and/or chronic T(3) effects on GLUT4 mRNA expression and polyadenylation, protein content, and trafficking to the plasma membrane (PM) in skeletal muscle, as well as on blood glucose disappearance rate (kITT) after insulin administration., Methods: Rats were surgically thyroidectomized and treated with T(3) (0.3 to 100 μg/100 g body weight) from 10 minutes to 5 days, and killed thereafter. Sham-operated (SO) rats were used as controls. Total RNA was extracted from the skeletal muscles (soleus [SOL] and extensorum digitalis longus [EDL]) and subjected to Northern blotting analysis using rat GLUT4 cDNA probe. Total protein was extracted and subjected to specific centrifugations for subcellular fractionation, and PM as well as microsomal (M) fractions were subjected to Western blotting analysis, using anti-GLUT4 antiserum as a probe. GLUT4 mRNA polyadenylation was examined by a rapid amplification of cDNA ends-poly(A) test (RACE-PAT)., Results: Thyroidectomy reduced skeletal muscle GLUT4 mRNA, mRNA poly(A) tail length, protein content, and trafficking to the PM, as well as the kITT. The acute T(3) treatment rapidly (30 minutes) increased all these parameters compared with Tx rats. The 5-day T(3) treatment increased GLUT4 mRNA and protein expression, and restored GLUT4 trafficking to the PM and kITT to SO values., Conclusions: The results presented here show for the first time that, in parallel to its transcriptional action on the SLC2A4 gene, T(3) exerts a rapid post-transcriptional effect on GLUT4 mRNA polyadenylation, which might increase transcript stability and translation efficiency, leading to the increased GLUT4 content and availability to skeletal muscle, as well as on GLUT4 translocation to the PM, improving the insulin sensitivity, as shown by the kITT.

Background: Cardiac arrest resuscitation can present myocardial dysfunction determined by ischemic time, and inhibition of the angiotensin-converting enzyme (ACE) can reduce cardiac dysfunction during reperfusion., Objective: To investigate the effects of angiotensin-I and different periods of ischemia on functional recovery in isolated rat hearts., Methods: Isolated hearts from Wistar rats (n=45; 250-300 g) were submitted to different periods of global ischemia (20, 25 or 30 min) and reperfused (30 min) with Krebs-Henseleit buffer alone or with the addition of 400 nmol/L angiotensin-I, or 400 nmol/L angiotensin-I + 100 μmol/L captopril along the reperfusion period., Results: The maximal positive derivative of pressure (+dP/dt(max)) and rate-pressure product were reduced in hearts exposed to 25 min ischemia (~73%) and 30 min ischemia (~80%) vs. 20 min ischemia. Left ventricular end-diastolic pressure (LVEDP) and perfusion pressure (PP) were increased in hearts exposed to 25 min ischemia (5.5 and 1.08 fold, respectively) and 30 min ischemia (6 and 1.10 fold, respectively) vs. 20 min ischemia. Angiotensin-I caused a decrease in +dP/dt(max) and rate-pressure product (~85-94%) in all ischemic periods and an increase in LVEDP and PP (6.9 and 1.25 fold, respectively) only at 20 min ischemia. Captopril was able to partially or completely reverse the effects of angiotensin-I on functional recovery in 20 min and 25 min ischemia., Conclusion: These data suggest that angiotensin-II directly or indirectly participates in the post-ischemic damage, and the ability of an ACE inhibitor to attenuate this damage depends on ischemic time.

Insulin replacement is the only effective therapy to manage hyperglycemia in type 1 diabetes mellitus (T1DM). Nevertheless, intensive insulin therapy has inadvertently led to insulin resistance. This study investigates mechanisms involved in the insulin resistance induced by hyperinsulinization. Wistar rats were rendered diabetic by alloxan injection, and 2 weeks later received saline or different doses of neutral protamine Hagedorn insulin (1.5, 3, 6, and 9 U/day) over 7 days. Insulinopenic-untreated rats and 6U- and 9U-treated rats developed insulin resistance, whereas 3U-treated rats revealed the highest grade of insulin sensitivity, but did not achieve good glycemic control as 6U- and 9U-treated rats did. This insulin sensitivity profile was in agreement with glucose transporter 4 expression and translocation in skeletal muscle, and insulin signaling, phosphoenolpyruvate carboxykinase/glucose-6-phosphatase expression and glycogen storage in the liver. Under the expectation that insulin resistance develops in hyperinsulinized diabetic patients, we believe insulin sensitizer approaches should be considered in treating T1DM.

Background: The effects of renal denervation on cardiovascular reflexes and markers of nephropathy in diabetic-hypertensive rats have not yet been explored., Aim: To evaluate the effects of renal denervation on nephropathy development mechanisms (blood pressure, cardiovascular autonomic changes, renal GLUT2) in diabetic-hypertensive rats. Forty-one male spontaneously hypertensive rats (SHR) ~250 g were injected with STZ or not; 30 days later, surgical renal denervation (RD) or sham procedure was performed; 15 days later, glycemia and albuminuria (ELISA) were evaluated. Catheters were implanted into the femoral artery to evaluate arterial pressure (AP) and heart rate variability (spectral analysis) one day later in conscious animals. Animals were killed, kidneys removed, and cortical renal GLUT2 quantified (Western blotting)., Results: Higher glycemia (p < 0.05) and lower mean AP were observed in diabetics vs. nondiabetics (p < 0.05). Heart rate was higher in renal-denervated hypertensive and lower in diabetic-hypertensive rats (384.8 +/- 37, 431.3+/- 36, 316.2 +/- 5, 363.8 +/- 12 bpm in SHR, RD-SHR,STZ-SHR and RD-STZ-SHR, respectively). Heart rate variability was higher in renal-denervated diabetic hypertensive rats (69.84 ± 37.91, 55.75 ± 25.21, 73.40 ±53.30, 148.4 ± 93 in SHR, RD-SHR, STZ-SHR- and RDSTZ-SHR, respectively, p < 0.05), as well as the LF component of AP variability (5.17 ± 5.24, 1.62 ± 0.9, 2.12 ±0.9, 7.38 ± 6.5 in SHR, RD-SHR, STZ-SHR and RDSTZ-SHR, respectively, p < 0.05). GLUT2 renal content was higher in all groups vs. SHR [corrected]., Conclusions: Renal denervation in diabetic-hypertensive rats improved previously reduced heart rate variability. The GLUT2 equally overexpressed by diabetes and renal denervation may represent a maximal derangement effect of each condition.

The effects of exercise training on systolic blood pressure (BP), insulin sensitivity, and plasma membrane GLUT4 protein content in spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats were compared. 16 SHR and 16 WKY male rats, aged 6 months, were randomized into sedentary and trained (treadmill running, 5 days/week, 60 min/day for 10 weeks) groups (n=8/group). At baseline, SHR had lower insulin sensitivity than WKY rats, however, there were no differences between WKY and SHR GLUT4 expression. The 10-week training reduced BP by ∼19% in SHR, improved insulin sensitivity by ∼24% in SHR, but not in WKY, and increased GLUT4 expression in both animal models. Compared to the sedentary group, there was an increase of GLUT4 in WKY rats by ∼25% in the heart, by ∼23% in the gastrocnemius, and by ∼15% in the fat tissue. Trained SHR presented an increase in GLUT4 of ∼21%, ∼20%, and ∼14%, in the same tissues, respectively. There were no differences between SHR and WKY rats in post-training GLUT4 expression. We conclude that training determined BP and insulin resistance reduction in SHR, and increased GLUT4 expression in both normotensive and hypertensive rats. However, considering the similar rise in GLUT4-induced training in SHR and WKY, it is possible that GLUT4 levels in plasma membrane fraction do not have a pivotal role in the exercise-induced improvement of insulin sensitivity in SHR., (© Georg Thieme Verlag KG Stuttgart · New York.)

Objective: To determine if resistive exercise protocol can modulate Tnf-α, SOCS3 and glucose transporter GLUT4 genes expression in skeletal muscle, and peripheral insulin sensitivity in obese rats induced by hyperlipidic diet., Materials and Methods: Wistar obese rats induced by hyperlipidic diet were subjected a resistive exercise protocol as jump squat. Insulin sensitivity and mRNA content of Tnf-α, SOCS3 and GLUT4 were assayed and compared among the groups: obese sedentary (OS) and exercised (OE), control sedentary (CS) and exercised (CE)., Results: The mRNA content of Tnf-α and SOCS3 has increased in skeletal muscle from OS and has decreased in OE group. The protein and GLUT4 mRNA contents were correlated but they did not change among the groups. Peripheral insulin sensitivity has increased in the OE compared to OS group., Conclusion: The resistive exercise reverses the peripheral insulin resistance and the inflammatory state in skeletal muscle from diet-induced obese rats.

Aim: Changes in skeletal muscle morphology and metabolism are associated with limited functional capacity in heart failure, which can be attenuated by neuromuscular electrical stimulation (ES). The purpose of the present study was to analyse the effects of ES upon GLUT-4 protein content, fibre structure and vessel density of the skeletal muscle in a rat model of HF subsequent to myocardial infarction., Methods: Forty-four male Wistar rats were assigned to one of four groups: sham (S), sham submitted to ES (S+ES), heart failure (HF) and heart failure submitted to ES (HF+ES). The rats in the ES groups were submitted to ES of the left leg during 20 days (2.5 kHz, once a day, 30 min, duty cycle 50%- 15 s contraction/15 s rest). After this period, the left tibialis anterior muscle was collected from all the rats for analysis., Results: HF+ES rats showed lower values of lung congestion when compared with HF rats (P = 0.0001). Although muscle weight was lower in HF rats than in the S group, thus indicating hypotrophy, 20 days of ES led to their recovery (P < 0.0001). In both groups submitted to ES, there was an increase in muscle vessel density (P < 0.04). Additionally, heart failure determined a 49% reduction in GLUT-4 protein content (P < 0.03), which was recovered by ES (P < 0.01)., Conclusion: In heart failure, ES improves morphological changes and raises GLUT-4 content in skeletal muscle., (© 2010 The Authors. Acta Physiologica © 2010 Scandinavian Physiological Society.)

The effect of glucose on the intracellular pH (pH(i)) recovery rate (dpH(i)/dt) and Na(+)-glucose transporter (SGLT) localization was investigated in HEK-293 cells, a cell line that expresses endogenous NHE1, NHE3, SGLT1, and SGLT2 proteins. The activity of the Na(+)/H(+) exchangers (NHEs) was evaluated by using fluorescence microscopy. The total and membrane protein expression levels were analyzed by immunoblotting. In cells cultivated in 5 mM glucose, the pH(i) recovery rate was 0.169 ± 0.020 (n = 6). This value did not change in response to the acute presence of glucose at 2 or 10 mM, but decreased with 25 mM glucose, an effect that was not observed with 25 mM mannitol. Conversely, the chronic effect of high glucose (25 mM) increased the pH(i) recovery rate (~40%, P < 0.05), without changes in the total levels of NHE1, NHE3, or SGLT1 expression, but increasing the total cellular (~50%, P < 0.05) and the plasma membrane (~100%, P < 0.01) content of SGLT2. Treatment with H-89 (10(-6) M) prevented the stimulatory effect of chronic glucose treatment on the pH(i) recovery rate and SGLT2 expression in the plasma membrane. Our results indicate that the effect of chronic treatment with a high glucose concentration is associated with increased NHEs activity and plasma membrane expression of SGLT2 in a protein kinase A-dependent way. The present results reveal mechanisms of glucotoxicity and may contribute to understanding the diabetes-induced damage of this renal epithelial cell.

Objective: To evaluate the transepithelial transport of sodium, glucose, potassium, and water and the mRNA level of the sodium-glucose cotransporter (SGLT1) and the facilitated sugar transporter (GLUT2) in the small intestine of iron-deficient rats., Methods: After 6 wk of receiving diets with low or normal iron content, rats (Wistar-EPM) were subjected to two experiments: 1) evaluation of the transepithelial transport of sodium, glucose, potassium, and water by an "in vivo" experimental model of intestinal perfusion and 2) determination of relative SGLT1 and GLUT2 mRNA levels in the proximal, intermediate, and distal portions of the small intestine by the northern blotting technique., Results: Hemoglobin and hepatic iron levels were statistically lower in the anemic rats. The mean transepithelial transports of sodium (-33.0 μEq · min(-1) · cm(-1), glucose (426.0 μM · min(-1) · cm(-1), and water (0.4 μL · min(-1) · cm(-1) in the small intestine of the anemic rats were significantly lower than in the control group (349.1 μEq · min(-1) · cm(-1), 842.6 μM · min(-1) · cm(-1), and 4.3 μl · min(-1) · cm(-1), respectively, P < 0.05). The transepithelial transport of potassium was similar for both groups. The relative SGLT1 mRNA levels of the anemic rats in the intermediate (1.796 ± 0.659 AU) and distal (1.901 ± 0.766 AU) segments were significantly higher than the values for the control rats (intermediate 1.262 ± 0.450 AU, distal 1.244 ± 0.407 AU). No significant difference was observed for the relative SLGT1 mRNA levels in the proximal segment or for the GLUT2 mRNA levels in all segments., Conclusion: Iron deficiency decreases the absorption of glucose, sodium, and water and increases SGLT1 mRNA in the intermediate and distal segments of the small intestine of rats., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Salivary gland dysfunction is a feature in diabetes and hypertension. We hypothesized that sodium-glucose cotransporter 1 (SGLT1) participates in salivary dysfunctions through a sympathetic- and protein kinase A (PKA)-mediated pathway. In Wistar-Kyoto (WKY), diabetic WKY (WKY-D), spontaneously hypertensive (SHR), and diabetic SHR (SHR-D) rats, PKA/SGLT1 proteins were analyzed in parotid and submandibular glands, and the sympathetic nerve activity (SNA) to the glands was monitored. Basal SNA was threefold higher in SHR (P < 0.001 vs. WKY), and diabetes decreased this activity (∼50%, P < 0.05) in both WKY and SHR. The catalytic subunit of PKA and the plasma membrane SGLT1 content in acinar cells were regulated in parallel to the SNA. Electrical stimulation of the sympathetic branch to salivary glands increased (∼30%, P < 0.05) PKA and SGLT1 expression. Immunohistochemical analysis confirmed the observed regulations of SGLT1, revealing its location in basolateral membrane of acinar cells. Taken together, our results show highly coordinated regulation of sympathetic activity upon PKA activity and plasma membrane SGLT1 content in salivary glands. Furthermore, the present findings show that diabetic- and/or hypertensive-induced changes in the sympathetic activity correlate with changes in SGLT1 expression in basolateral membrane of acinar cells, which can participate in the salivary glands dysfunctions reported by patients with these pathologies.